Cell-mediated autoimmunity has been suggested to be involved in the melanocyte apoptosis that occurs in vitiligo. We investigated the cytotoxicity to autologous melanocytes of CD8+ T cells from the perilesional margins and peripheral blood samples of vitiligo patients. CD8+ T cells isolated from skin biopsied from the edges of depigmented skin patches of vitiligo patients or from peripheral blood samples of the same donors were proliferated in culture medium. The primary cultures of CD8+ T cells and autologous melanocytes were mixed at ratios of 1:1, 1:2 or 1:5 and incubated for 3 days. The apoptosis of the melanocytes was analyzed by flow cytometry. Secreted cytokines in selected samples were measured by cytokine arrays. The results show that the CD8+ T cells were successfully isolated from the vitiligo perilesional margins. This cell population showed a significantly higher percentage of CD69 expression (56.13±3.55 versus 29.93±2.35%, p<0.01) and CD137 expression (41.74±1.06 versus 25.97±1.63%, p<0.01) compared with CD8+ T cells in peripheral blood from the same donors. The co-culturing of CD8+ T cells from lesional skin with autologous melanocytes induced apoptosis in the melanocytes (16.63±1.21, 16.71±0.63 and 18.32±1.60% for CD8+ T cells and autologous melanocytes at ratios of 1:1, 1:2 and 1:5, respectively). IL-6 levels were much higher in the co-culture (3.01-fold higher than in a melanocyte monoculture and 17.32-fold higher than in a CD8+ T-cell monoculture). The CD8+ T cells were also demonstrated to secrete more IL-13. Taken together, our data demonstrate that the infiltration of active CD8+ T cells takes place in the vitiligo perilesional margins. Those CD8+ T cells present significantly higher activation levels and higher cytotoxicity to autologous melanocytes than their counterparts from peripheral blood samples. These data suggest that CD8+ T cells are likely to be involved in the pathogenesis of vitiligo.

We here investigated the efficiency of autologous melanocyte transplantation of 23 vitiligo patients by focusing on perilesional skin homing CD8+ T lymphocytes, and studied the potential effect of dermal mesenchymal stem cells (DMSCs) on CD8+ T cell activities in vitro. Out of 23 patients with the autologous melanocyte transplantation, 12 patients (52.17%) had an excellent re-pigmentation, 6 patients (26.09%) had a good re-pigmentation, 5 patients (21.74%) had a fair or poor re-pigmentation. CD8+ T cells infiltrating was observed in the perilesional vitiligo area of all patients. Importantly, the efficiency of the transplantation was closely associated with skin-homing CD8+ T cell activities. The patients with high number of perilesional CD8+ T cells or high level of cytokines/chemokines were associated with poor re-pigmentation efficiency. For in-vitro experiments, we successfully isolated and characterized human DMSCs and skin-homing CD8+ T cells. We established DMSCs and CD8+ T cell co-culture system, where DMSCs possessed significant inhibitory effects against skin homing CD8+ T lymphocytes. DMSCs inhibited CD8+ T cells proliferation, induced them apoptosis and regulated their cytokines/chemokines production. Our results suggest that vitiligo patients’ autologous melanocytes transplantation efficiency might be predicted by perilesional skin-homing CD8+ T cell activities, and DMSCs might be used as auxiliary agent to improve transplantation efficacy.

Autologous minigrafts with ultrapulsed carbon dioxide laser abrasion appear to have the potential to achieve complete repigmentation in patients with stable vitiligo who fail to respond to medical treatments.